N2 non-thermal atmospheric pressure plasma promotes wound healing in vitro and in vivo: Potential modulation of adhesion molecules and matrix metalloproteinase-9.

Advances in physics and biology have made it possible to apply non-thermal atmospheric pressure plasma (NTP) in the biomedical field. Although accumulating evidence suggests that NTP has various medicinal effects, such as facilitating skin wound healing on exposed tissue while minimizing undesirable tissue damage, the underlying molecular mechanisms are not fully understood. In this study, NTP generated from N2 optimized wound healing in the scratch wound healing assay. In addition, matrix metalloproteinase (MMP)-9 expression and enzyme activity increased and the urokinase-type plasminogen activator (uPA) system was activated after NTP treatment. We also showed that NTP treatment increased Slug and TCF8/ZEB1 expression and decreased that of E-cadherin, suggesting induction of the epithelial-to-mesenchymal transition (EMT). The effect of N2 NTP was verified on rat wound model. Taken together, these results suggest that N2 NTP promotes wound healing by inducing the EMT and activating the MMP-9/uPA system. These findings show the therapeutic potential of NTP for skin wound healing.

Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae.

Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death.

Non-thermal atmospheric pressure plasma induces apoptosis in oral cavity squamous cell carcinoma: Involvement of DNA-damage-triggering sub-G(1) arrest via the ATM/p53 pathway.

Recent advances in physics have made possible the use of non-thermal atmospheric pressure plasma (NTP) in cancer research. Although increasing evidence suggests that NTP induces death of various cancer cell types, thus offering a promising alternative treatment, the mechanism of its therapeutic effect is little understood. In this study, we report for the first time that NTP led to apoptotic cell death in oral cavity squamous cell carcinoma (OSCC). Interestingly, NTP induced a sub-G(1) arrest in p53 wild-type OSCCs, but not in p53-mutated OSCCs. In addition, NTP increased the expression levels of ATM, p53 (Ser 15, 20 and 46), p21, and cyclin D1. A comet assay, Western blotting and immunocytochemistry of γH2AX suggested that NTP-induced apoptosis and sub-G(1) arrest were associated with DNA damage and the ATM/p53 signaling pathway in SCC25 cells. Moreover, ATM knockdown using siRNA attenuated the effect of NTP on cell death, sub-G(1) arrest and related signals. Taken together, these results indicate that NTP induced apoptotic cell death in p53 wild-type OSCCs through a novel mechanism involving DNA damage and triggering of sub-G(1) arrest via the ATM/p53 pathway. These findings show the therapeutic potential of NTP in OSCC.

Chip-based cartilage oligomeric matrix protein detection in serum and synovial fluid for osteoarthritis diagnosis.

We have developed a method to detect cartilage oligomeric matrix protein (COMP) as a specific biomarker of osteoarthritis (OA). In pathological conditions of the cartilage, COMP is released first into the synovial fluid (SF) and from there into the blood. Thus, measurement of COMP in the blood and SF facilitates OA diagnosis. To determine COMP, we developed a fluoro-microbead guiding chip (FMGC)-based immunoassay. The FMGC has four immunoreactive regions, each with five patterns, to allow multiple assays. A COMP-specific capture antibody was immobilized to the FMGC surface to create a self-assembled interfacial layer. SF or serum samples from patients with OA possessing the target COMP were applied to the COMP-sensing monolayer. To generate binding signal, COMP detection antibody-conjugated fluoro-microbeads were applied and the numbers of fluoro-microbeads bound specifically were counted to determine COMP concentrations. This FMGC-based immunoassay clearly distinguished immunospecific from nonspecific binding by comparing optical signals from inside and outside of the patterns. The optical signals showed linear correlations with serum and SF COMP concentrations. Optical detection and quantification of COMP using fluorescence microscopy correlated well with results from commercial enzyme-linked immunosorbent assay (ELISA). This FMGC-based immunoassay offers a new approach for detecting a clinically relevant biomarker for OA in human blood and SF.

Development of chipless, wireless current sensor system based on giant magnetoimpedance magnetic sensor and surface acoustic wave transponder.

A chipless, wireless current sensor system was developed using a giant magnetoimpedance (GMI) magnetic sensor and one-port surface acoustic wave (SAW) reflective delay line for real-time power monitoring in a current-carrying conductor. The GMI sensor has a high-quality crystalline structure in each layer, which contributes to a high sensitivity and good linearity in a magnetic field of 3-16 Oe. A 400 MHz RF energy generated from the interdigital transducer (IDT)-type reflector on the one-port SAW delay line was used as an activation source for the GMI magnetic sensor. The one-port SAW delay line replaces the presently existing transceiver system, which is composed of thousands of transistors, thus enabling chipless and wireless operation. We confirmed a large variation in the amplitude of the SAW reflection peak with a change in the impedance of the GMI sensor caused by the current flow through the conductor. Good linearity and sensitivity of ~0.691 dB/A were observed for currents in the range 1-12 A. Coupling of Mode (COM) modeling and impedance matching analysis were also performed to predict the device performance in advance and these were compared with the experimental results.

A pneumatic micro cell chip for the differentiation of human mesenchymal stem cells under mechanical stimulation.

A new micro cell chip which can induce stem cells to differentiate into specific body cell types has been designed and fabricated for tissue engineering. This paper presents the test results of a micro cell stimulator which can provide a new miniaturized tool in cell stimulation, culture and analysis for stem cell research. The micro cell stimulator is designed to apply compressive pressure to the hMSCs (human mesenchymal stem cells) for inducing osteogenesis. The micro cell stimulator is based on the pneumatic actuator with a flexible diaphragm which consists of an air chamber and cell chambers. The hMSCs under cyclic compressive stimulation for one week were observed and assessed by monitoring CD90 (Thy-1), actin, alkaline phosphatase (ALP) and alizarin red expression. The results suggest that cyclic mechanical stimulation is attributed to the different phenomenon of cultured hMSCs in cell proliferation and differentiation. These results are important for the feasibility of the micro cell stimulator to provide the reduction of the necessary quantity of cells, process cost and the increase of the throughput.

Comparative Effects of Non-Thermal Atmospheric Pressure Plasma on Migration and Invasion in Oral Squamous Cell Cancer, by Gas Type.

PURPOSE: The fourth state of matter, plasma is known as an ionized gas with electrons, radicals and ions. The use of non-thermal plasma (NTP) in cancer research became possible because of the progresses in plasma medicine. Previous studies on the potential NTP-mediated cancer therapy have mainly concentrated on cancer cell apoptosis. In the present study, we compared the inhibitory effect of NTP on cell migration and invasion in the oral squamous cancer cell lines. MATERIALS AND METHODS: We used oral squamous cancer cell lines (SCC1483, MSKQLL1) and different gases (N2, He, and Ar). To investigate the mechanism of plasma treatment, using different gases (N2, He, and Ar) which induces anti-migration and anti-invasion properties, we performed wound healing assay, invasion assay and gelatin zymography. RESULTS: The results showed that NTP inhibits cancer cell migration and invasion of oral squamous cancer cell. In addition, focal adhesion kinase expression and matrix metalloproteinase-2/9 activity were also inhibited. CONCLUSION: The suppression of cancer cell invasion by NTP varied depending on the type of gas. Comparison of the three gases revealed that N2 NTP inhibited cell migration and invasion most potently via decreased expression of focal adhesion kinase and matrix metalloproteinase activity.

An electromagnetic compressive force by cell exciter stimulates chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

In this study, we present a biological micro-electromechanical system and its application to the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs). Actuated by an electromagnetic force, the micro cell exciter was designed to deliver a cyclic compressive load (CCL) with various magnitudes. Two major parts in the system are an actuator and a cartridge-type chamber. The former has a permanent magnet and coil, and the latter is equipped with 7 sample dishes and 7 metal caps. Mixed with a 2.4% alginate solution, the alginate/MSC layers were positioned in the sample dishes; the caps contained chondrogenic defined medium without transforming growth factor-beta (TGF-beta). Once powered, the actuator coil-derived electromagnetic force pulled the metal caps down, compressing the samples. The cyclic load was given at 1-Hz frequency for 10 min twice a day. Samples in the dishes without a cap served as a control. The samples were analyzed at 3, 5, and 7 days after stimulation for cell viability, biochemical assays, histologic features, immunohistochemistry, and gene expression of the chondrogenic markers. Applied to the alginate/MSC layer, the CCL system enhanced the synthesis of cartilage-specific matrix proteins and the chondrogenic markers, such as aggrecan, type II collagen, and Sox9. We found that the micromechanically exerted CCL by the cell exciter was very effective in enhancing the chondrogenic differentiation of MSCs, even without using exogenous TGF-beta.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-alpha1-antichymotrypsin detection based on surface plasmon resonance.

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-alpha(1)-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.

Anti-cancer efficacy of nonthermal plasma dissolved in a liquid, liquid plasma in heterogeneous cancer cells.

The therapeutic potential of nonthermal plasma for cancer treatment has been reported recently. The heterogeneity of cancer cells need to be addressed to design effective anticancer treatments. Here, we show that treatment with nonthermal atmospheric-pressure plasma dissolved in a liquid (liquid plasma) induces oxidative stress in heterogeneous populations of cancer cells and ultimately kills these cells via apoptosis, regardless of genetic status, e.g., mutations in p53 and other DNA-damage-response genes. We found that liquid plasma markedly increased the concentration of intracellular and mitochondrial reactive oxygen species (ROS), reflecting an influx from the extracellular milieu. Liquid plasma contributed to mitochondrial accumulation of ROS and depolarization of mitochondrial membrane potential with consequent cell death. Healthy normal cells, however, were hardly affected by the liquid-plasma treatment. The antioxidant N-acetylcysteine blocked liquid-plasma-induced cell death. A knockdown of CuZn-superoxide dismutase or Mn-SOD enhanced the plasma-induced cell death, whereas expression of exogenous CuZn-SOD, Mn-SOD, or catalase blocked the cell death. These results suggest that the mitochondrial dysfunction mediated by ROS production is a key contributor to liquid-plasma-induced apoptotic cell death, regardless of genetic variation. Thus, liquid plasma may have clinical applications, e.g., the development of therapeutic strategies and prevention of disease progression despite tumor heterogeneity.

Novel Therapeutic Effects of Non-thermal atmospheric pressure plasma for Muscle Regeneration and Differentiation.

Skeletal muscle can repair muscle tissue damage, but significant loss of muscle tissue or its long-lasting chronic degeneration makes injured skeletal muscle tissue difficult to restore. It has been demonstrated that non-thermal atmospheric pressure plasma (NTP) can be used in many biological areas including regenerative medicine. Therefore, we determined whether NTP, as a non-contact biological external stimulator that generates biological catalyzers, can induce regeneration of injured muscle without biomaterials. Treatment with NTP in the defected muscle of a Sprague Dawley (SD) rat increased the number of proliferating muscle cells 7 days after plasma treatment (dapt) and rapidly induced formation of muscle tissue and muscle cell differentiation at 14 dapt. In addition, in vitro experiments also showed that NTP could induce muscle cell proliferation and differentiation of human muscle cells. Taken together, our results demonstrated that NTP promotes restoration of muscle defects through control of cell proliferation and differentiation without biological or structural supporters, suggesting that NTP has the potential for use in muscle tissue engineering and regenerative therapies.

Detection of CTX-II in serum and urine to diagnose osteoarthritis by using a fluoro-microbeads guiding chip.

This study reports a new strategy for simultaneous detection of the C-telopeptide fragments of type II collagen (CTX-II) as a biomarker of osteoarthritis (OA) using a fluoro-microbeads guiding chip. As osteoarthritis progresses, the joint components including matrix and cartilage are degraded by proteases. The degraded products such as CTX-II are released into the serum and urine, and the CTX-II concentration in body fluids reflects OA progression. Because the CTX-II has heterogeneous epitope structure in serum (sCTX-II; homodimers) and urine (uCTX-II; monomers or variant monomers), a multiple-sensing device enabling both sandwich and competitive-type immunoassays is required. For multiple assessments of serum and urinary CTX-II, we designed a fluoro-microbeads guiding chip (FMGC) containing multiple sensing areas and connecting channels. Using the approach, the sandwich (sCTX-II) and competition (uCTX-II) assays could be simultaneously performed on a single chip. We designed a fluidic control device enabling selective control of the open-close function of FMGC channels. The immune-specific signal was quantitatively analyzed by counting the number of fluorescent microbeads from the registered images. The results from the developed FMGC assay showed high correlation with those obtained in ELISA. The completion time of the FMGC assay was 24-fold and 3.5-fold shorter than the ELISA for urinary and serum CTX-II. Taken together, it enabled the simultaneous detection of both sCTX-II and uCTX-II. This FMGC-based assay would be a promising tool for monitoring of osteoarthritis.

Combination of NTP with cetuximab inhibited invasion/migration of cetuximab-resistant OSCC cells: Involvement of NF-κB signaling.

Although the epidermal growth factor receptor (EGFR) is an established target in head-and-neck cancer (HNC), resistance to EGFR-targeted therapy mediated by various mechanisms has been reported. Therefore, a combination strategy to overcome resistance to EGFR mono-targeted therapy is clinically required. We have previously demonstrated that non-thermal atmospheric pressure plasma (NTP) induces death of various cancer cells, including oral squamous cancer (OSCC) cells. In this study, we report for the first time that combining NTP treatment with cetuximab led to inhibition of migration and invasion in cetuximab-resistant OSCC cells, which could be a promising strategy to overcome resistance to anti-EGFR therapy. NTP induced deactivation of NF-κB in SCCQLL1 cells, but not in MSKQLL1 cells. In addition, NTP increased the expression level of E-cadherin, and decreased those of vimentin, Slug, Snail, matrix metalloproteinase (MMP)-2, -9, and activities of MMPs. Moreover, NF-κB upregulation using cDNA diminished the combination effect of NTP on invasion, migration and related signals. Taken together, these results indicate that the combination of NTP with cetuximab can decrease invasiveness in cetuximab-resistant OSCCs through a novel mechanism involving the NF-κB pathway. These findings show the therapeutic potential of treatment that combines NTP and cetuximab in OSCC.

Targeting cancer cells with reactive oxygen and nitrogen species generated by atmospheric-pressure air plasma.

The plasma jet has been proposed as a novel therapeutic method for cancer. Anticancer activity of plasma has been reported to involve mitochondrial dysfunction. However, what constituents generated by plasma is linked to this anticancer process and its mechanism of action remain unclear. Here, we report that the therapeutic effects of air plasma result from generation of reactive oxygen/nitrogen species (ROS/RNS) including H2O2, Ox, OH-, •O2, NOx, leading to depolarization of mitochondrial membrane potential and mitochondrial ROS accumulation. Simultaneously, ROS/RNS activate c-Jun NH2-terminal kinase (JNK) and p38 kinase. As a consequence, treatment with air plasma jets induces apoptotic death in human cervical cancer HeLa cells. Pretreatment of the cells with antioxidants, JNK and p38 inhibitors, or JNK and p38 siRNA abrogates the depolarization of mitochondrial membrane potential and impairs the air plasma-induced apoptotic cell death, suggesting that the ROS/RNS generated by plasma trigger signaling pathways involving JNK and p38 and promote mitochondrial perturbation, leading to apoptosis. Therefore, administration of air plasma may be a feasible strategy to eliminate cancer cells.

Nonthermal plasma induces apoptosis in ATC cells: involvement of JNK and p38 MAPK-dependent ROS.

PURPOSE: To determine the effects of nonthermal plasma (NTP) induced by helium (He) alone or He plus oxygen (O2) on the generation of reactive oxygen species (ROS) and cell death in anaplastic thyroid cancer cells. MATERIALS AND METHODS: NTP was generated in He alone or He plus O2 blowing through a nozzle by applying a high alternating current voltage to the discharge electrodes. Optical emission spectroscopy was used to identify various excited plasma species. The apoptotic effect of NTP on the anaplastic thyroid cancer cell lines, such as HTH83, U-HTH 7, and SW1763, was verified with annexin V/propidium staining and TUNEL assay. ROS formation after NTP treatment was identified with fluorescence-activated cell sorting with DCFDA staining. The mitogen-activated protein kinase pathways and caspase cascade were investigated to evaluate the molecular mechanism involved and cellular targets of plasma. RESULTS: NTP induced significant apoptosis in all three cancer cell lines. The plasma using He and O2 generated more O2-related species, and increased apoptosis and intracellular ROS formation compared with the plasma using He alone. NTP treatment of SW1763 increased the expression of phosphor-JNK, phosphor-p38, and caspase-3, but not phosphor-ERK. Apoptosis of SW1763 as well as expressions of elevated phosphor-JNK, phosphor-p38, and caspase-3 induced by NTP were effectively inhibited by intracellular ROS scavengers. CONCLUSION: NTP using He plus O2 induced significant apoptosis in anaplastic cancer cell lines through intracellular ROS formation. This may represent a new promising treatment modality for this highly lethal disease.

Non-thermal atmospheric pressure plasma inhibits thyroid papillary cancer cell invasion via cytoskeletal modulation, altered MMP-2/-9/uPA activity.

Plasma, the fourth state of matter, is defined as a partially or completely ionized gas that includes a mixture of electrons and ions. Advances in plasma physics have made it possible to use non-thermal atmospheric pressure plasma (NTP) in cancer research. However, previous studies have focused mainly on apoptotic cancer cell death mediated by NTP as a potential cancer therapy. In this study, we investigated the effect of NTP on invasion or metastasis, as well as the mechanism by which plasma induces anti-migration and anti-invasion properties in human thyroid papillary cancer cell lines (BHP10-3 and TPC1). Wound healing, pull-down, and Transwell assays demonstrated that NTP reduced cell migration and invasion. In addition, NTP induced morphological changes and cytoskeletal rearrangements, as detected by scanning electron microscopy and immunocytochemistry. We also examined matrix metalloproteinase (MMP)-2/-9 and urokinase-type plasminogen activator (uPA) activity using gelatin zymography, uPA assays and RT-PCR. FAK, Src, and paxillin expression was detected using Western blot analyses and immunocytochemistry. NTP decreased FAK, Src, and paxillin expression as well as MMP/uPA activity. In conclusion, NTP inhibited the invasion and metastasis of BHP10-3 and TPC1 cells by decreasing MMP-2/-9 and uPA activities and rearranging the cytoskeleton, which is regulated by the FAK/Src complex. These findings suggest novel actions for NTP and may aid in the development of new therapeutic strategies for locally invasive and metastatic cancers.

Chip-based comparison of the osteogenesis of human bone marrow- and adipose tissue-derived mesenchymal stem cells under mechanical stimulation.

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (ß1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.

Atmospheric-pressure plasma jet induces apoptosis involving mitochondria via generation of free radicals.

The plasma jet has been proposed as a novel therapeutic method for anticancer treatment. However, its biological effects and mechanism of action remain elusive. Here, we investigated its cell death effects and underlying molecular mechanisms, using air and N2 plasma jets from a micro nozzle array. Treatment with air or N2 plasma jets caused apoptotic death in human cervical cancer HeLa cells, simultaneously with depolarization of mitochondrial membrane potential. In addition, the plasma jets were able to generate reactive oxygen species (ROS), which function as surrogate apoptotic signals by targeting the mitochondrial membrane potential. Antioxidants or caspase inhibitors ameliorated the apoptotic cell death induced by the air and N2 plasma jets, suggesting that the plasma jet may generate ROS as a proapoptotic cue, thus initiating mitochondria-mediated apoptosis. Taken together, our data suggest the potential employment of plasma jets as a novel therapy for cancer.

A fluoro-microbead guiding chip for simple and quantifiable immunoassay of cardiac troponin I (cTnI).

We have developed a fluoro-microbead guiding chip (FMGC) to perform an optical immunoassay of cardiac troponin I (cTnI). The plasma marker protein cTnI is the currently preferred marker to use for a definitive diagnosis and prognosis of myocardial infarction. The FMGC has four immunoreaction regions on a silicon oxide substrate, with five gold patterns imprinted on each region for multiple simultaneous assays. The FMGC assay clearly distinguished immunospecific binding from nonspecific binding by comparing optical signals from inside and outside of the patterns. To detect cTnI, a sandwich immunoassay was performed using antibody-tagged fluoro-microbeads. The cTnI-specific capture antibody was conjugated to the FMGC surface by reaction with 3-3'-dithiobis-propionic acid N-hydroxysuccinimide ester to create a self-assembling antigen-sensing monolayer (DTSP SAM) on the chip. A sample containing cTnI was applied to the antigen-sensing monolayer and allowed to react. To generate a binding signal, a cTnI detection antibody-linked fluoro-microbead preparation was added. The cTnI concentration in a sample was determined by counting the number of biospecifically bound fluoro-microbeads on the corresponding five patterns on the FMGC. The optical signal showed a linear correlation with cTnI concentrations in plasma samples containing from 3.4 pM to 3.4 nM (0.1-100 ng/ml) cTnI. The sensitivity of cTnI detection could be increased by reducing the non-specific binding of the beads to the antigen-sensing surfaces of the chip. Optical detection and quantification of binding by fluorescence microscopy gave results that correlated well with results from a commercial ELISA for cTnI in human plasma. Based on these findings, we propose that the FMGC-based immunoassay system may be adapted to detect and quantify a variety of clinically important targets in human samples.

Fabrication and testing of a PDMS multi-stacked hand-operated LOC for use in portable immunosensing systems.

This paper presents the development of a reliable multi-liquid lab-on-a-chip (LOC), with a hand-operated mechanism, for the application in portable immunosensing systems. To control the transport of multiple liquids without any external equipment, we utilize capillary attraction force for filling and surface tension for stopping liquid flow. As a driving force, hydraulic pressure caused by the elastic deformation of a liquid reservoir transfers liquid stopped at passive valves. The proposed LOC successfully demonstrates a reliable sequential liquid transfer within the reaction channel. To highlight its feasibility as a portable diagnostic system, we performed the electrochemical immunoassay measuring antibody concentrations within the fabricated LOC. As a test biorecognition reaction, the anti-dinitrophenyl (DNP) antibody with an enzymatic catalysis was selected as the target analyte. The amplified signals obtained from this experiment indicated a high selectivity of the immunosensing LOC.